We would like to thank the members of the Potter group for providing invaluable comments on the protocol. This work was funded by a clinical research training fellowship from the American Academy of Neurology Foundation to CMH, a fellowship from the National Institute of General Medical Sciences (NIGMS; <a href="http://www.nigms.nih.gov/">http://www.nigms.nih.gov/</a>) to JDR (GM08169), a grant from the NIH National Institute of Neurological Disorders and Stroke (NS054809), a Ruth L. Kirschstein National Research Service Award to JDR (NS060392), an NSF EFRI grant (0836017), the Coulter Foundation Translational Research Award, and a translational research fellowship to JDR (NS007480).

<ol>
	<li>Potter, S. M., DeMarse, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+neural+cell+culture+for+long-term+studies.">A new approach to neural cell culture for long-term studies.</a> J Neurosci Methods. 110, 1-2 (2001).</li><li>Rambani, K., Vukasinovic, J., Glezer, A., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+thick+brain+slices:+an+interstitial+3D+microperfusion+system+for+enhanced+viability.">Culturing thick brain slices: an interstitial 3D microperfusion system for enhanced viability.</a> J Neurosci Methods. 180, 243-254 (2009).</li><li>Bakkum, D. J., Chao, Z. C., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatio-temporal+electrical+stimuli+shape+behavior+of+an+embodied+cortical+network+in+a+goal-directed+learning+task.">Spatio-temporal electrical stimuli shape behavior of an embodied cortical network in a goal-directed learning task.</a> J Neural Eng. 5, 310-323 (2008).</li><li>Shahaf, G., Marom, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+in+networks+of+cortical+neurons.">Learning in networks of cortical neurons.</a> J Neurosci. 21, 8782-8788 (2001).</li><li>Wagenaar, D. A., Nadasdy, Z., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+dynamic+attractors+in+activity+patterns+of+cultured+neuronal+networks.">Persistent dynamic attractors in activity patterns of cultured neuronal networks.</a> Phys Rev E Stat Nonlin Soft Matter Phys. 73, 051907-05 (2006).</li><li>Esposti, F., Signorini, M. G., Potter, S. M., Cerutti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+long-term+correlations+in+dissociated+cortical+neuron+recordings.">Statistical long-term correlations in dissociated cortical neuron recordings.</a> IEEE Trans Neural Syst Rehabil Eng. 17, 364-369 (2009).</li><li>Madhavan, R., Chao, Z. C., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+recurring+spatiotemporal+activity+patterns+in+cortical+networks.">Plasticity of recurring spatiotemporal activity patterns in cortical networks.</a> Phys Biol. 4, 181-193 (2007).</li><li>Chiappalone, M., Massobrio, P., Martinoia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network+plasticity+in+cortical+assemblies.">Network plasticity in cortical assemblies.</a> Eur J Neurosci. 28, 221-237 (2008).</li><li>Hofmann, F., Bading, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+term+recordings+with+microelectrode+arrays:+studies+of+transcription-dependent+neuronal+plasticity+and+axonal+regeneration.">Long term recordings with microelectrode arrays: studies of transcription-dependent neuronal plasticity and axonal regeneration.</a> J Physiol Paris. 99, 2-3 (2006).</li><li>Gortz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+reduction+of+spontaneous+neuronal+network+activity+by+sublethal+amyloid+beta+(1-42)+peptide+concentrations.">Transient reduction of spontaneous neuronal network activity by sublethal amyloid beta (1-42) peptide concentrations.</a> J Neural Transm. 116, 351-355 (2009).</li><li>Koito, H., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+rat+brain+aggregate+cultures+for+neuron+and+glia+development+studies.">Preparation of rat brain aggregate cultures for neuron and glia development studies.</a> J Vis Exp. 31, (2009).</li><li>Brewer, G. J., Cotman, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+growth+of+hippocampal+neurons+in+defined+medium+at+low+density:+advantages+of+a+sandwich+culture+technique+or+low+oxygen.">Survival and growth of hippocampal neurons in defined medium at low density: advantages of a sandwich culture technique or low oxygen.</a> Brain Res. 494, 65-74 (1989).</li><li>Lelong, I. H., Petegnief, V., Rebel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+cells+mature+faster+on+polyethyleneimine+coated+plates+than+on+polylysine+coated+plates.">Neuronal cells mature faster on polyethyleneimine coated plates than on polylysine coated plates.</a> J Neurosci Res. 32, 562-568 (1992).</li><li>Potter, S. M., Pine, J., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+transplant+staining+with+DiI+and+vital+imaging+by+2-photon+laser-scanning+microscopy.">Neural transplant staining with DiI and vital imaging by 2-photon laser-scanning microscopy.</a> Scanning Microsc Suppl. 10, 189-199 (1996).</li><li>Rolston, J. D., Gross, R. E., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+low-cost+multielectrode+system+for+data+acquisition+enabling+real-time+closed-loop+processing+with+rapid+recovery+from+stimulation+artifacts.">A low-cost multielectrode system for data acquisition enabling real-time closed-loop processing with rapid recovery from stimulation artifacts.</a> Front Neuroengineering. 2, (2009).</li><li>Wagenaar, D. A., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+all-channel+stimulator+for+electrode+arrays,+with+real-time+control.">A versatile all-channel stimulator for electrode arrays, with real-time control.</a> J Neural Eng. 1, 39-45 (2004).</li><li>van Pelt, J., Wolters, P. S., Corner, M. A., Rutten, W. L., Ramakers, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+characterization+of+firing+dynamics+of+spontaneous+bursts+in+cultured+neural+networks.">Long-term characterization of firing dynamics of spontaneous bursts in cultured neural networks.</a> IEEE Trans Biomed Eng. 51, 2051-2062 (2004).</li><li>Wagenaar, D. A., Pine, J., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+extremely+rich+repertoire+of+bursting+patterns+during+the+development+of+cortical+cultures.">An extremely rich repertoire of bursting patterns during the development of cortical cultures.</a> BMC Neurosci. 7, (2006).</li><li>Wagenaar, D. A., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+multi-channel+stimulus+artifact+suppression+by+local+curve+fitting.">Real-time multi-channel stimulus artifact suppression by local curve fitting.</a> J Neurosci Methods. 120, 113-120 (2002).</li><li>Bakkum, D. J., Chao, Z. C., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+activity-dependent+plasticity+of+action+potential+propagation+delay+and+amplitude+in+cortical+networks.">Long-term activity-dependent plasticity of action potential propagation delay and amplitude in cortical networks.</a> PLoS One. 3 (5), e2088-e2088 (2008).</li><li>Wagenaar, D. A., Madhavan, R., Pine, J., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+bursting+in+cortical+cultures+with+closed-loop+multi-electrode+stimulation.">Controlling bursting in cortical cultures with closed-loop multi-electrode stimulation.</a> J Neurosci. 25 (3), 680-688 (2005).</li><li>Jimbo, Y., Tateno, T., Robinson, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+induction+of+pathway-specific+potentiation+and+depression+in+networks+of+cortical+neurons.">Simultaneous induction of pathway-specific potentiation and depression in networks of cortical neurons.</a> Biophys J. 76 (2), 670-678 (1999).</li><li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing nerve cells. , (1998).</li></ol>

CMH has nothing to disclose. 
JDR and SMP are paid consultants for Axion Biosystems. 
The submission fees and video processing fees for this article and video were graciously sponsored by Multi-Channel Systems.

This protocol shows instructions on how to culture and maintain neuronal networks on micro-electrode arrays as well as an introduction to recording from and stimulating neuronal networks. Some of the more common problems when recording from MEAs were also discussed in the MEA troubleshooting section. There are occasional variations in the protocol as discussed throughout and oftentimes these changes are utilized to optimize specific conditions required by certain experimental designs.
Although the recording and stimulating system presented here is comprised of our custom designed NeuroRighter15 with the MCS preamp, there are multiple other systems that can provide an interface with the neuronal networks. Choosing a particular setup will depend on the specific experimental needs.
Simplistic recording and stimulation examples were provided in this protocol however these cultures on MEAs can be used to provide insight into complex questions about synaptic activity and neuronal network connectivity. As mentioned above, ongoing work is utilizing this technology to study processes like cellular plasticity, development, excitotoxicity and neurodegeneration.6-10 For example, a recent publication from our laboratory showed reproducible goal directed learning in vitro with real-time closed loop stimulation in response to current neuronal network activity3.
Although MEA cultures are often finicky and delicate, thanks to their simplicity and accessibility (compared to intact animals), they provide a powerful model for studying neuronal activity at a network level.

Supplies
<ul>
 <li>Disposable bottle top filter (150ml, 0.2&mu;m pore size; Nalgene #565-0020)</li>
 <li>Embryonic day 18 timed-pregnant female rat</li>
 <li>Microelectrode arrays (see description in protocol, B1)</li>
 <li>Microelectrode array lids (see description in protocol, B3)</li>
 <li>Nylon cell strainer (40&mu;m, BD Falcon, #352340)</li>
 <li>Polypropylene conical vials (sterile, 15ml)</li>
 <li>Polystyrene Petri dishes (sterile, 35mm and 100x15mm)</li>
 <li>Pipet tips (sterile, 20&mu;l, 200&mu;l and 1000&mu;l)</li>
 <li>Serological pipets (5ml)</li>
</ul>
Equipment:
<ul>
 <li>Biological safety cabinet/laminar flow hood</li>
 <li>Cell counter</li>
 <li>Centrifuge</li>
 <li>Container of ice</li>
 <li>Dissecting scissors and forceps (sterilized)</li>
 <li>Dissecting microscope in a laminar flow hood</li>
 <li>Handheld electric pipetman </li>
 <li>Hemacytometer</li>
 <li>Isofluorane and gas chamber</li>
 <li>Light microscope</li>
 <li>Handheld pipetman (P-20 (20&mu;l), P-200 (200&mu;l), P-1000 (1000&mu;l))</li>
 <li>MEA recording system (see description in protocol, D1)</li>
 <li>Rodent Guillotine</li>
 <li>Tri-gas incubator (see description in protocol, B15)</li>
 <li>Vacuum flask with aspirator attached in the hood</li>
 <li>Vortex</li>
 <li>Water bath (35-37&deg;C)</li>
</ul>
Solutions and Reagents:
BSA solution: 
 
 Make 5% BSA (Sigma A-9418) in phosphate buffered saline pH 7.4 and filtered at 0.2&mu;m. This solution can be prepared ahead of time and stored at 4&deg;C for several months.
Cell Medium: 
 
 Combine 90 ml Dulbecco's modified Eagle's medium (Irvine Scientific 9024), 10 ml horse serum (Gibco 16050), 1ml sodium pyruvate (100mM, Gibco 11360), 250 &mu;l GlutaMAX (Gibco 35050), and insulin (final concentration 2.5 &mu;g/ml, Sigma I-5500) then pass through a 0.2 &mu;m filter.22 Warm and equilibrate the cell medium in the incubator prior to use. 
Dissection solution for preparing papain solution:23
<table border="1">
 <tr>
 <td>Dissection solution (100ml stock)</td>
 <td>Combine in order, filter 0.22 &mu;m and store at 4&deg;C.</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Component</td>
 <td>Volume ml</td>
 <td>Final Concentration (mM)</td>
 </tr>
 <tr>
 <td>Double-distilled water</td>
 <td>91.175</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1 M MgCl2</td>
 <td>0.58</td>
 <td>5.8 mM</td>
 </tr>
 <tr>
 <td>1 M CaCl2</td>
 <td>0.025</td>
 <td>0.25 mM</td>
 </tr>
 <tr>
 <td>0.5 M HEPES</td>
 <td>0.32</td>
 <td>1.6 mM</td>
 </tr>
 <tr>
 <td>0.5% Phenol red</td>
 <td>0.2</td>
 <td>(0.001%)</td>
 </tr>
 <tr>
 <td>0.1 N NaOH</td>
 <td>0.2</td>
 <td>0.2 mM</td>
 </tr>
 <tr>
 <td>2 M Na2SO4</td>
 <td>4.5</td>
 <td>90 mM</td>
 </tr>
 <tr>
 <td>1 M K2SO4</td>
 <td>3.0</td>
 <td>30 mM</td>
 </tr>
 <tr>
 <td>0.1 M Kynurenic acid</td>
 <td>1.0</td>
 <td>1 mM</td>
 </tr>
 <tr>
 <td>5 mM APV</td>
 <td>1.0</td>
 <td>0.05 mM</td>
 </tr>
</table>
This solution can be prepared ahead of time and stored at 4&deg;C for several months.
DNAse I: 
 
 Invitrogen (#18047019). 50&mu;l aliquots are flash frozen and stored at -80&deg;C. An aliquot is thawed in the laminar flow hood prior to starting the cell dissociation process. 
Hank's balanced salt solution: 
 
 Sigma (#4891). A 1X solution is prepared in deionized water, sterile filtered at 0.2&mu;m and stored at 4&deg;C.
Hibernate solution: 
 
 Prepare on day of culturing. Combine 98 ml L-15 medium (Invitrogen 11415) with 2 ml B-27 supplement (50X, Invitrogen #17504) and filter at 0.2 &mu;m.
Laminin solution: 
 
 Prepare on day of culturing. Dilute 20&mu;l of laminin (1mg/ml, Sigma L-2020) in 980 &mu;l cell medium for a final concentration of 0.02mg/ml. 20 &mu;l aliquots of the laminin (1mg/ml) are stored at -80&deg;C and thawed just prior to use. Thawing should occur on the bench top and not in the water bath as rapid thawing can sometimes lead to gelling of the laminin.
Papain solution:23 
 
 Warm 10 ml of dissection solution (above) to 30-32&deg;C. Add 200 units papain (Sigma P-4762) and 1.6 mg L-cysteine (Sigma C-7880). Adjust pH to 7.4 with about 14 &mu;l of 0.1 N NaOH. Incubate for about 30 minutes until the solution clears, filter 0.2 &mu;m, flash freeze with liquid nitrogen in 2 ml aliquots in 15 ml polypropylene sterile conical vials and store at -80&deg;C. Thaw an aliquot at 35-37&deg;C prior to starting protocol.
Polyethyleneimine (PEI) solution:13 
 
 100 &mu;l of PEI (50% w/v, Sigma P-3143) is diluted in 100 ml sodium borate buffer (0.1 M pH 8.4, Sigma B-9876) for a final concentration of 0.05% w/v. Sterile filter at 0.2 &mu;m. This can be prepared in advance and stored at 4&deg;C for months. 
Sterile de-ionized water: 
 
 Autoclave and allow to cool to room temperature prior to use. 
Trypsin (0.25% with EDTA, Gibco 25200): 
 
 1ml aliquots are flash frozen and stored at -80&deg;C. Aliquots are thawed in the 35-37&deg;C water bath prior to use.

how to culture, record and stimulate neuronal networks on micro-electrode arrays (meas)

For the last century, many neuroscientists around the world have dedicated their lives to understanding how neuronal networks work and why they stop working in various diseases. Studies have included neuropathological observation, fluorescent microscopy with genetic labeling, and intracellular recording in both dissociated neurons and slice preparations. This protocol discusses another technology, which involves growing dissociated neuronal cultures on micro-electrode arrays (also called multi-electrode arrays, MEAs).
There are multiple advantages to using this system over other technologies. Dissociated neuronal cultures on MEAs provide a simplified model in which network activity can be manipulated with electrical stimulation sequences through the array's multiple electrodes. Because the network is small, the impact of stimulation is limited to observable areas, which is not the case in intact preparations. The cells grow in a monolayer making changes in morphology easy to monitor with various imaging techniques. Finally, cultures on MEAs can survive for over a year in vitro which removes any clear time limitations inherent with other culturing techniques.1
Our lab and others around the globe are utilizing this technology to ask important questions about neuronal networks. The purpose of this protocol is to provide the necessary information for setting up, caring for, recording from and electrically stimulating cultures on MEAs. In vitro networks provide a means for asking physiologically relevant questions at the network and cellular levels leading to a better understanding of brain function and dysfunction.

Watch this Scientific Journal Video about How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays MEAs at JoVE.com

How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays MEAs

This protocol provides the necessary information for setting up, caring for, recording from and electrically stimulating cultures on MEAs. In vitro networks provide a means for asking physiologically relevant questions at the network and cellular levels leading to a better understanding of brain function and dysfunction.

How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays (MEAs)

For the last century, many neuroscientists around the world have dedicated their lives to understanding how neuronal networks work and why they stop ...

how-to-culture-record-stimulate-neuronal-networks-on-micro-electrode

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54.3K Views.  Emory University School of Medicine. This protocol provides the necessary information for setting up, caring for, recording from and electrically stimulating cultures on MEAs. In vitro networks provide a means for asking physiologically relevant questions at the network and cellular levels leading to a better understanding of brain function and dysfunction.

Video: How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays MEAs

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Techniques to Induce and Quantify Cellular Senescence

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed cellular senescence. Accumulation of senescent cells occurs with organismal aging and through continual culturing in vitro. Senescent cells influence many biological processes, including embryonic development, tissue repair and regeneration, tumor suppression, and aging. Hallmarks of senescent cells include, but are not limited to, increased senescence-associated &#946;-galactosidase activity (SA-&#946;-gal); p16INK4A, p53, and p21 levels; higher levels of DNA damage, including &#947;-H2AX; the formation of Senescence-associated Heterochromatin Foci (SAHF); and the acquisition of a Senescence-associated Secretory Phenotype (SASP), a phenomenon characterized by the secretion of a number of pro-inflammatory cytokines and signaling molecules. Here, we describe protocols for both replicative and DNA damage-induced senescence in cultured cells. In addition, we highlight techniques to monitor the senescent phenotype using several senescence-associated markers, including SA-&#946;-gal, &#947;-H2AX and SAHF staining, and to quantify protein and mRNA levels of cell cycle regulators and SASP factors. These methods can be applied to the assessment of senescence in various models and tissues.

Cellular senescence, the irreversible state of cell-cycle arrest, can be induced by various cellular stresses. Here, we describe protocols to induce cellular senescence and methods to assess markers of senescence.

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed ...

An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights the need to better understand the molecular mechanism involved in adipocyte dysfunction to develop new therapies against obesity-related diseases. Modulating the expression of proteins and micro-RNAs in adipocytes remains highly challenging. This paper describes a protocol to differentiate murine fibroblasts into mature adipocytes and to modulate the expression of proteins and micro-RNAs in mature adipocytes through reverse-transfection using small-interfering RNA (siRNA) and micro-RNA mimicking (miR mimic) oligonucleotides. This reverse-transfection protocol involves the incubation of the transfection reagent and the oligonucleotides to form a complex in the cell culture plate to which the mature adipocytes are added. The adipocytes are then allowed to reattach to the adherent plate surface in the presence of the oligonucleotides/transfection reagent complex. Functional analyses such as the study of insulin signaling, glucose uptake, lipogenesis, and lipolysis can be performed on the transfected 3T3-L1 mature adipocytes to study the impact of protein or micro-RNA manipulation on adipocyte function.

Presented here is a protocol to deliver oligonucleotides such as small-interfering RNA (siRNA), micro-RNA mimics (miRs), or anti-micro-RNA (anti-miR) into mature adipocytes to modulate protein and micro-RNA expression.

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...